ResearchArticle
Received: 16November 2010 Revised: 18 February 2011 Accepted: 5 March 2011 Published onlinein Wiley OnlineLibrary:
(wileyonlinelibrary.com) DOI 10.1002/ps.2183
Foliarresiduedynamicsofazadirachtinsfollowingdirectsteminjectionintowhiteandgreenashtreesforcontrolofemeraldashborer
SusanaGrimalt,
a
DeanThompson,
a
∗
DerekChartrand,
a
JohnMcFarlane,
a
BlairHelson,
a
BarryLyons,
a
JoeMeating
b
andTaylorScarr
c
Abstract
BACKGROUND: Azadirachtins are natural insecticides derived from the neem tree. The emerald ash borer (EAB) is an exoticinvasiveinsectpestthatinfestsvariousashtreespeciesandhasthepotentialforsignificanteconomic,aestheticandecologicalimpacts throughout North America. The initial translocation and foliar residue dynamics of azadirachtins were examinedfollowingdirectinjectionintowhiteandgreenashtreesgrowinginurbanscenariosasapotentialcontrolforEAB.RESULTS:SubstantialconcentrationsofazadirachtinsAandB[meanmaxima
>
0
.
98mgkg
−
1
freshweight(f.w.)]wereobservedwithin 2days of injecting a specifically designed formulation of azadirachtins. Foliar residues declined exponentially throughtime, with half-life estimates ranging from 5.1 to 12.3days. At the time of leaf senescence, foliar residue levels approximated0.01mgkg
−
1
f.w.,stronglymitigatingthepotentialeffectsofnon-targetbiotainsoiloraquaticcompartments.CONCLUSION:Themagnitudeanddurationofexposuresobservedinthisfieldstudywereconsideredtobeabovethethresholdsrequired for biological effectiveness against both larval and adult life stages of EAB. Results support the use of azadirachtinsas an environmentally acceptable systemic insecticide for control of EAB and protection of high-value ash trees in urbanenvironments.c
2011SocietyofChemicalIndustryKeywords:
azadirachtin; uptake; dissipation; systemic injection; emerald ash borer
1 INTRODUCTION
A recentreview
1
estimated 360cases of alien insect species intro-ductions into North American forests, with attendant economiclosses approximating $2.1 billion. Meyerson and Mooney
2
notedthat invasive pest introductions are very likely to increase in con-cert with the process of commercial globalization and increasinginternational trade. The Canadian Government Strategy on Inva-sive Species
3
also emphasized the alarming increase in the rateof introductions of invasive alien species and noted that they arenow considered the second greatest threat to biodiversity afterhabitat loss. Langor
etal
.
4
documented more than 419 exotic in-sects and mites occurring on woody plants in Canada. As notedby Haack,
5
many exotic insects feed on trees and shrubs, withseveral recent arrivals threatening to spread across continentalscales.Amongthelattergroupofinvasives,theemeraldashborer(
Agrilusplanipennis
)stands out as a key problem species. Theemeraldashborer(EAB)wasfirstdiscoveredinNorthAmer-ica near Detroit, Michigan, in 2002, and has since become estab-lished in 15 differentUSstates (http://www.aphis.usda.gov/planthealth/plant pest info/emerald ash b/downloads/multistateeab.pdf),aswellasthroughoutsouthernOntarioandintotheprovinceof Quebec in Canada (http://www.inspection.gc.ca/english/plaveg/pestrava/agrpla/regrestrice.shtml). While research hasdemonstrated that EAB infestations are not likely to developon tree species other than ash,
6
surveys have indicated that allnativeashspeciesaresusceptible.
7
Greenashtreesarereportedlycolonized earlier and succumb more rapidly than white ash wheninfested by EAB on the same site.
7
In the environs of the originalinfestations in the Great Lakes watershed, several species of ash,particularly green and white ash (
Fraxinus pennsylvanica
) and(
Fraxinus americana
), were extensively planted as shade trees inparks and streetboulevards, as well as to replaceelm(
Ulmus
spp.)which had been previously devastated by Dutch elm disease.
8
Ash was also planted in numerous marginal agricultural landreclamation projects in southern Ontario. In some cases, these
∗
Correspondence to: DeanThompson, Canadian Forest Service, Natural Resources Canada, 1219 Queen St East, Sault Ste. Marie, Ontario P6A 2E5,Canada.E-mail:dthompso@nrcan.gc.ca
a
CanadianForest Service,NaturalResources Canada, SaultSte. Marie, Ontario,Canada
b
BioForestTechnologiesInc.,SaultSte.Marie,Ontario,Canada
c
OntarioMinistryofNaturalResources,ForestResourcesGroup,SaultSte.Marie,Ontario,Canada
PestManagSci
(2011) www.soci.orgc
2011 Society of Chemical Industry
www.soci.org S Grimalt
etal
.and other species of ash also representsignificant components of remnantnaturalforests,woodlotsandriparianzonesinthisarea.
9
As noted by previous authors,
10
EAB adults feed on ashfoliage particularly during the early summer to acquire energyfor maturation and breeding; however, adults cause relativelylittle damage to the tree per se. Typically, leaf feeding occurs fora period of approximately 2 weeks before mating and ovipositionbegin.EggsarelaidinbarkcrevicesofthemainstemandbranchesofthetreefromlateJunetoearlyAugustandhatchwithinroughly2 weeks. Larvae chew through the bark and feed on phloem untillateautumn,therebyweakeningthetree.Successiveyearsoflarvalfeedingactivityeffectivelyresultingirdlingofthetree,preventingsufficient uptake and translocation of water and nutrients andultimately tree mortality.Given the abundance of ash trees in both urban and remnantnatural forests throughout northeastern North America, theirgeneric susceptibility to EAB attack and the invasive characterof EAB, there is an exceedingly high risk of economic, ecologicalandaestheticimpacts.
10
Aneffectiveintegratedpestmanagementapproachisthereforerequiredtomitigatethoserisks.Whileseveralparasitoid insect species and microbial agents are currently beinginvestigated as potential biocontrols,
7,11,12
none has been shownto provide control over field populations to date. Meanwhile,EAB-infested areas increase dramatically with each passing year,and thus there is an immediate need for non-destructive controloptions with the ability to protect ash trees of high economic,aestheticorecologicalvalue.IntheUnitedStates,severalchemicalcontrol techniques employing various application methods andsystemic insecticides have been developed and are being used.Principal among these is the application of systemic insecticidessuch as imidacloprid or emamectin benzoate using either steminjection,soilinjectionorsoildrenchtechniques.
13
Althoughsomeof these options have been shown to result in high levels of adultmortality and significant reductions in larval density, there aresubstantialenvironmentalconcernsassociatedwithsomeofthesecompoundsandapplicationtechniques.Inparticular,soilinjectionorsoildrenchtechniquesmayresultinunnecessaryexposureandrisk to non-target organisms in both the soil and potentiallyadjacent aquatic compartments.
14,15
In general, environmentalconcerns are exacerbated when the compounds involved arerelatively persistent, and such issues may significantly influenceregistration or operational use in some jurisdictions.In Canada, where major EAB infestations often occur inassociation with urban environments, legislative bans, as well asgeneral public opposition to the use of conventional insecticides,significantly constrain potential chemical control options. Giventhat the treatment objectives in urban scenarios revolve aroundprotection of relatively small numbers of high-value ash trees,single-tree application techniques are viable options. Among thevariety of potential techniques available, direct stem injectionsusing insecticides with a favorable environmental profile areconsidered most likely to receive registration and be acceptedby the general public.Azadirachtins are a family of natural tetranortriterpenoidcompounds of botanical origin. They are particularly prevalentin seeds of the neem tree (
Azadirachta indica
Juss.) and exhibit avariety of exploitable biological activities, including significantantifeedant, antifertility and growth-regulating properties ininsects.
16
Neem seed extracts are particularly rich in two closelyrelatedcompounds,referredtoasazadirachtinsAandB(Aza-AandAza-B), that are considered to be the putative active ingredientsforobservedeffectsoninsects.Formulationspreparedfromtheseextracts have activity on a variety of wood-boring and foliarpests and varying levels of systemic activity.
17–20
Azadiracthinsalso exhibit relatively low toxicity to mammals, birds and non-target invertebrates.
21,22
A recent review on the environmentalfate and effects of azadirachtins in relation to potential uses inCanadian forestry
23
demonstrated that formulations containingazadirachtins have low to moderate persistence in water, soiland foliage and generally do not present a significant risk to non-targetspecies,withtheexceptionofsomefreshwaterzooplanktonspecies. Given their general activity against wood-boring insectpests, systemic properties, low mammalian toxicity and positiveenvironmental profile, azadirachtins are particularly well suitedfor development and use as a potential control of invasive wood-boring insect pests such as EAB.In a recently published paper by the present authors,
24
proof of concept was provided through the demonstration of dramaticinhibitionoflarvalgrowthanddevelopment,reductionsinfeedinggalleriesandultimatemortalityofEABlarvaeatrelativelylowdoselevels in small and large green ash following stem injections withthe formulated product TreeAzin
. In the present paper, thispotential is examined further by comparatively assessing foliarresiduedynamicsinbothgreenandwhiteashtreesgrowingundertypical urban parkland and street boulevard scenarios typical of anticipated primary use patterns.
2 EXPERIMENTALMETHODS
2.1 Experimentaldesign,siteandtreecharacterization
The experimental site chosen for this study was located in thevicinity of Carriage Hill Park, London, Ontario. The approximatesite coordinates are (NAD 83 UTM 17T 4776814763209). Thegeneral site location, as well as the specific coordinates foreach experimental tree, was obtained using a handheld GarminGPSmap76CXx GPS unit (Garmin International Inc., Olathe, KS). Atthetimeofstudyinitiationinthesummerof2007,thegeneralareawas a known site of EAB infestation and being managed underquarantine restrictions. A few ash trees in the immediate areashowed symptoms of latter-stage EAB infestation, including bark cracking, characteristic D-shaped exit holes and crown dieback.WithintheparkareaandalongtheboulevardofnearbyMasonvilleCrescent, a total of 13 ash trees of similar total height, mainstem diameter and dimension of live crown canopy (Table 1)were selected and considered as replicate experimental units.Experimental trees were specifically selected for uniformity, tobe free of physical damage on the main stem and as havinghealthy canopies with no evidence of crown dieback or chloroticfoliage.Stemdiameteratbreastheight(dbh)wasmeasuredwithastandarddbhtape.Measurementsoftreeheight,aswellasheightand diameter of live crown, were made using a laser measuringdevice (LaserAce 300;MDL, Houston, TX). The study was designed to compare white and green ash treesgrowing in the park setting (
n
=
4 for each species), with an addi-tionalseparatecomparisonofresiduedynamicsingreenashtrees(
n
=
5) growing in park or street boulevard conditions. As therewere no white ash trees growing in nearby street boulevards, asimilar comparisonamongwhite ashin the two differentgrowingenvironments was not possible. Hence, the experiment may beconsidered as havingthree groupsorclasses (i.e. greenash – park setting, greenash – streetboulevardsetting, whiteash – parkset-ting).Meantreemeasurementsbyspeciesandgrowingconditionclass are given in Table 1. Comparative climatic data describinggrowingconditions (temperature and rainfall)in the year of study
wileyonlinelibrary.com/journal/ps
c
2011 Society of Chemical Industry
PestManagSci
(2011)
Azadirachtin follar residue dynamics in relation to emerald ash borer control www.soci.org
Table1.
Characterization data for white (
FraxinusAmericana
) and green (
Fraxinuspennsylvanica
) ash trees selectedfor this experiment
a
Tree dbh (cm) Tree height(m) Canopy height (m) Canopy diameter(m)Species Class Mean CV
b
(%) Mean CV (%) Mean CV (%) Mean CV (%)Green ash Park 19 6 9 11 7 11 6 21White ash Park 19 15 9 8 7 25 6 29Green ash Boulevard 21 10 10 3 8 11 7 7
a
ANOVA indicatednosignificant differencesamong means for tree diameterat breast height(dbh),tree height, heightof live canopyor diameterof live canopy(
P
>
0
.
10).
b
CV: coefficientof variation.
Total monthly precipitation
T o t a l P r e c i p i t a t i o n ( m m )
020406080100120
AprilMayJuneJulyAugSept
(a)
Monthly mean temperartureMonths of the Year
M e a n T e m p e r a t u r e ( C )
051015202510 Year Avg (1997-2006)Experimental Year (2007)AprilMayJuneJulyAugSept
(b)
Figure1.
Monthlytotalprecipitation(a) andmeanairtemperatures(b) forthe experimental year (2007), as compared with the 10 year average forthe period (1999–2006).
versus averages for the preceding 10 years for this area (Fig. 1)were extracted from the National Climate and Information DataArchive (http://www.climate.weatheroffice.gc.ca/) for the LondonInternational Airport and London CS stations.
2.2 Steminjectionmethods
TreeAzin
systemic insecticide is a proprietary formulation of azadirachtins owned by the Canadian Forest Service and licensedworldwide for distribution by BioForest Technologies Inc. Theproduct is exempt from the current Cosmetic Pesticide BanlegislationwhichisnowinplaceintheprovinceofOntario,whereit is scheduled as a class 4 or ‘low-risk’ pesticide, and is also listedby the Organic Materials Review Institute for use in organic crops. TreeAzin
contains50 mg mL
−
1
or5%totalazadirachtinsA
+
Basthe active ingredients. The formulation was prepared on 19 June2007,usingtechnical,activepowdermaterialprovidedbyEIDParry(Bangalore,India).Thecertifiedtotalazadirachtins(A
+
B)contentoflot138usedtopreparetheformulationtestedinthisstudy was42%, a value verified as accurate on the basis of an independentanalysis via a liquid chromatography diode array detector (LC-DAD). Following preparation of the TreeAzin
formulation (lot2007-1), analysis of a subsample showed a concentration of 4
.
6
µ
g mL
−
1
or 92% of the expected total azadirachtin (A
+
B)content. The ratio of active ingredients (Aza-A:Aza-B) in the finalformulation was approximately 75:25%.Inthisstudy,allexperimentaltreeswereinjectedwithTreeAzin
on26June 2007atarateof0.2 gAIcm
−
1
dbh(equivalentto 4 mLof TreeAzin
per cm dbh) under an experimental use permit(48-RP-07) granted by the Pest Management Regulatory Agencyof Health Canada. Stem injections were made with the EcoJect
system for direct stem injection of systemic pesticides, which wasrecentlyintroducedbyBioForestTechnologiesInc,SaultSte.Marie,ON. Stem injections were made following general instructionsas documented in the operating manual
23
but employing fourinjection ports per tree as a standard protocol. The creation of injection ports involved drilling four 0.58cm (15/64 inch) holesat equal spacings about the circumference of the main stem atapproximately15–30cmabovetheground.Eachholewasdrilledto a depth of 1.3–1.9 cm (0.5–0.75 inches) into the xylem tissueat a downward angle of 20–45
◦
. A plastic EcoJect
nozzle wastheninsertedsnuglyintoeachportandmatedtoan8 mLcanisterthat had been prefilled with the TreeAzin
formulation using theEcoJect
pump system. Once attached to the nozzle, the spring-loaded system automatically injected the formulation into thetree. Details pertaining to the stem injections for each tree areprovided in Table 2, and an example of the stem injection set-upas employed in this study is shown in Fig. 2. Given the injectionvolumes required, a total of eight full canisters were routinelyapplied to each tree (two canisters deployed in sequence at eachport). Residual volumes required to attain the total volume to beinjected for any individual tree were subsequently applied usingmeasured volumes in partially filled canisters.
2.3 Foliarsampling
Composite samples of canopy foliage were obtained from eachexperimental tree at several discrete time periods. Sampling wasinitiated on 28 June 2007, 2 days after treatment (DAT), andthereafter at 7, 14, 29, 43, 56, 70 and 83 DAT as well as at 365
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